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Nuclear Medicine and Biology 37 (2010) 197 – 204www.elsevier.com/locate/nucmedbio

Uptake of 3-[125 I]iodo-α-methyl-L-tyrosine into colon cancer DLD-1 cells:
characterization and inhibitory effect of natural amino acids andamino acid-like drugs☆
Naoto Shikanoa,⁎, Masato Oguraa , Hiroyuki Okudairab , Syuichi Nakajimaa , Takashi Kotania ,Masato Kobayashib , Shinya Nakazawaa , Takeshi Babac , Naoto Yamaguchic , Nobuo Kubotaa ,Yukio Iwamurad , Keiichi KawaibaDepartment of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japanb

School of Health Sciences, Faculty of Medicine, Kanazawa University, Kanazawa 920-0942, Japanc

Center for Medical Sciences, Ibaraki Prefectural University of Health Sciences, Ami-machi, Inashiki-gun, Ibaraki 300-0394, JapandCenter for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, Ami-machi, Inashiki-gun, Ibaraki 300-0394, Japan
Received 20 June 2007; received in revised form 7 October 2009; accepted 31 October 2009

Abstract
Introduction: We examined 3-[123I]iodo-α-methyl-L-tyrosine ([123I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in
the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure.
Methods: Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [125I]
IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs(1 mM) on [125I]IMT uptake were examined.
Results: Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected;
system L (LAT3, LAT4) and MCT8 were weakly detected; and B0AT was not detected. [125I]IMT uptake in DLD-1 cells involved
Na+-independent system L primarily and Na+-dependent system(s). Uptake of [125I]IMT in Na+-free buffer followed Michaelis–Menten
kinetics, with a Km of 78 μM and Vmax of 333 pmol/106 cells per minute. Neutral D- and L-amino acids with branched or aromatic large side
chains inhibited [125I]IMT uptake. Tyrosine analogues, tryptophan analogues, L-phenylalanine and p-halogeno-L-phenylalanines, and gamma
amino acids [including 3,4-dihydroxy-L-phenylalanine (L-DOPA), DL-threo-β-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]-L-phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [125I]IMT uptake, but L-tyrosine methyl ester andR(+)/S(−)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [125I]IMT uptake except L-serine andD/L-cysteine.
Conclusions: [125I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived fromtyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in
LAT1 depends on the structure of the group corresponding to the amino acid residue. Beta-hydroxylation may confer reduction of transport
affinity of tyrosine analogues via LAT1.
© 2010 Elsevier Inc. All rights reserved.
Keywords: Amino acid-like drug; Colon cancer cell line; DLD-1; 3-Iodo-α-methyl-L-tyrosine; Inhibitor; L-Type amino acid tranasporter-1☆
This work was supported by Grants-in-Aid for Scientific Research (#10770451, #14770498, #13557075, #15659283, #16659322 and #17390336) from
the Ministry of Education, Science, Sports and Culture of Japan and the Japan Society for the Promotion of Science. Financial support was also provided by the
Ibaraki Prefectural University Research Project (9808, 0118, and 0220); Ibaraki Prefectural University Grants-in-Aid of the Encouragement for Young Scientists
2001, 2002, 2004, 2005, 2006, 2008 and 2009; and Japan Atherosclerosis Research Foundation Grant-2008.
⁎ Corresponding author. Tel.: +81 29 840 2217; fax: +81 29 840 2317.
E-mail address: sikano@ipu.ac.jp (N. Shikano).

0969-8051/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2009.10.011

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N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 197–204

1. Introduction
3-[123I]Iodo-α-methyl-L-tyrosine ([123I]IMT), an artificial
amino acid, has been developed as a functional imaging
agent for tyrosine transport mechanisms in the brain andpancreas [1–3]. Because amino acids rapidly accumulate intumor cells for active proliferation, [123 I]IMT has also beenused clinically for SPECT imaging of tumors, as describedby Langen et al. [4–8].
Compared to intracranial tumor cell lines including rat C6
glioma cells [9], human GOS3 glioma cells [10] and 86HG39 human glioma cells [8], relatively few of the studies of[123I]IMT transport have involved extracranial tumor celllines. The kinetics of [123 I]IMT transport have been studiedin human Ewing's sarcoma cells [11]. Furthermore, [123I]
IMT transport has been characterized in rat lymphoma cells[12], a porcine kidney epithelial cell line [13], humanmonocyte-macrophages [14], human small-cell lung cancer[15] and pancreatic carcinoma [16].
In cultured glioma cells of the lines mentioned above,
membrane transport of [123I]IMT is dominated by BCHsensitive transport, i.e., amino acid transport system L, andrelatively minor uptake occurs via the Na+-dependent system[8–10]. System T also mediates [123 I]IMT transport into
U266 human myeloma cells [17]. However, the gene
expression of neutral amino acid transporters has not been
fully clarified even in those cell lines.
Some of the system L transporters demonstrate gene
expression inducement that is dependent on the cellularconditions, and thus researchers in the fields of cell
activation, cellular proliferation and cellular nutrition havebecome interested in the inhibitors and/or substrates of thesystem L transporter family [18–20]. The results of suchstudies are applied to not only cancer imaging [7] ortherapeutics [21], but also to the development of a new classof target in immunosuppressant studies [22].
In the present study, we characterized [125I]IMT transport
in the human colon cancer cell line, DLD-1, with particularemphasis on the inhibitory effect of amino acid-like drugs.
The gene expression of neutral amino acid transporters
was confirmed with real-time reverse transcription–PCR(real-time PCR) in DLD-1. We selected the following aminoacid-like drugs for investigation: DL-threo-β-(3,4-dihydroxyphenyl)serine [DOPS; amino acid precursor of noradrenaline (NA); elevates brain NA concentrations] [23], 4-[bis(2-chloroethyl)amino]-L-phenylalanine (melphalan; antineoplastic agent that forms DNA intrastrand crosslinks
by bifunctional alkylation in 5′-GGC sequences) [21],β-(aminomethyl)4-chlorobenzenepropanoic acid [R(+)baclofen; GABAB receptor agonist: more active enantiomer;
skeletal muscle relaxant, S(−)-baclofen and less active
enantiomer of baclofen] [24], 1-(aminomethyl)-cyclohexaneacetic acid (gabapentin; anticonvulsant with unknown
mechanism of action, crosses the blood–brain barrier,increases GABA concentrations in the brain and reduces
excitatory amino acid neurotransmission) [25], and others aslisted in Materials and Methods. In addition, we discuss thecorrelation between the inhibition effect and the structure ofthese compounds. To the best of our knowledge, there are no
previously published data on the inhibitory effects of suchamino acid analogues on [125 I]IMT transport.
2. Materials and methods
2.1. Materials and preparation of labeled compounds
Reagent-grade chemicals were acquired from SigmaAldrich Japan KK (Tokyo, Japan). Instead of 123 I (T1/2=13 h)for clinical use, 125 I-labeled IMT ([125I]IMT) was used forconvenience because of its longer half-life (T1/2=60 days).
[125 I]NaI (8.1×107 GBq/mmol) was purchased from Muromachi Chemical Co. (Tokyo, Japan). Human colorectal
adenocarcinoma cell line DLD-1 was obtained from theJapanese Collection of Research Bioresources (Tokyo,
Japan; catalog number JCRB9094). Plastic tissue culture
dishes (diameter, 60 mm; Nalge Nunc International Roskilde,
Denmark) and plastic flasks (surface area, 25 cm2; Nalge
Nunc International Roskilde) were used. No-carrier-added[125 I]IMT was prepared using the conventional chloramine-T
method, as described elsewhere [1–3,13]. [125 I]IMT labelingefficiency and radiochemical purities were greater than 80%
and 95%, respectively. Specific radioactivity was greater than
8.1×1019 Bq/mol.
2.2. Cell cultures
DLD-1 cells were maintained in cells fed with Dulbecco's
Modified Eagle's Medium (D-MEM; Sigma-Aldrich Japan)
supplemented with 10% FBS and 2 mM glutamine, in a 5%
CO2 humidified atmosphere at 37°C. For the uptake
experiments, DLD-1 cells were seeded on 60-mm dishes ata cell density of 5×105 cells/dish in 5 ml of complete medium
and were used on the third or fourth day after inoculation.
2.3. Measurement of [125I]IMT transport in DLD-1 cells
For the uptake study, we investigated the time course of[125 I]IMT uptake (incubation time: 5, 10, 30, 45 and 60 min),contributions of transport systems [in sodium-free medium
or sodium-containing medium and sodium-free medium
containing 1 mM of 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH)], concentration dependence (at 3, 10, 30,
100 and 300 mM of IMT) and the effects of natural aminoacids (1 mM) on [125I]IMT uptake. In addition, we examined
natural amino acid and amino acid-like drug (1 mM)
inhibition of [125I]IMT transport into DLD-1 cells in Na+containing uptake medium. Kinetic parameters with [125I]
IMT uptake were determined based on the results of theconcentration-dependence study.
The sodium-containing medium was phosphate-bufferedsaline, pH 7.4, which was composed of 137 mM NaCl,
2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM K2HPO4, CaCl2
1.8 mM and MgCl 1 mM. In the sodium-free experiment,
NaCl and Na2HPO4 were replaced with the same concentrations of choline-Cl and K2HPO4, respectively. After removal

N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 197–204

of the culture medium, each dish was washed once with 5 mlof incubation medium for 10 min at 37°C. The cells werethen incubated with 2 ml of incubation medium containing
18.5 kBq [125 I]IMT for various periods of time at 37°C.
For the inhibition experiment, one of several inhibitors wasadded, and the cells were then incubated for 10 min at 37°C
with 18.5 kBq [125 I]IMT. The following inhibitors were
tested at 1.0 mM: 20 D- or L-amino acids, BCH, R(+)baclofen, S(−)-baclofen, 1-(aminomethyl)-cyclohexaneacetic acid (gabapentin), 3,4-dihydroxy- L -phenylalanine(L-DOPA), DOPS, 4-[bis(2-chloroethyl)amino]-L-phenylalanine (melphalan), seleno-L-methionine and 2-(methylamino)isobutyric acid (MeAIB); N-methylglycine (sarcosine).
After incubation, the medium was aspirated, and themonolayers were rinsed twice rapidly with 5 ml of ice-cold
incubation medium. The cells were solubilized in 1.5 ml of
1N NaOH, and the radioactivity of each aliquot wasquantified using a well-type scintillation counter.
2.4. RNA extraction and quantitative real-time reverse
transcription–PCR
Total RNA from DLD-1 cells was isolated using the
QIAshredder and RNeasy Plus Mini Kit (QIAGEN)
according to the manufacturer's description on the third or
fourth day after inoculation. cDNA was synthesized using
the Transcriptor First Strand cDNA Synthesis Kit (Roche
Applied Science, Mannheim, Germany), using random
hexamers priming at 50°C for 1 h, according to the
manufacturer's instructions. The primers (Table 1) were
designed using the Roche Applied Science website-based
Universal ProbeLibrary Assay Design Center (http://www.
roche-applied-science.com). Quantitative real-time PCR was
performed using LightCycler 2.0 instruments, LightCycler
TaqMan Master and the Universal ProbeLibrary (Roche
Applied Science) with the following profile: one cycle at
95°C for 10 min, 45 cycles of 95°C for 10 s and 60°C for
25 s, and, finally, one cycle at 40°C for 30 s. PCR products
were analyzed by agarose gel electrophoresis, and no199

nonspecific PCR bands were detected. The relative levelsof PCR products were calculated from standard curves
established from each primer pair. Expression data werenormalized against the amount of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) and actin beta(ACTB) PCR
products as the housekeeping genes [26,27].
2.5. Statistical analysis
Data represent the mean±S.D. of five measurements, andeach experiment was performed in duplicate. Results wereanalyzed using Student's t test. The Pb.01 level was
considered to denote statistical significance.

3. Results
A representative growth curve of DLD-1 is shown in
Fig. 1A. DLD-1 cells 3 to 4 days after inoculation were
semiconfluent logarithmic phase cells and were used inthis experiment.
Fig. 1B shows the time course of [125I]IMT uptake into
DLD-1 cells. Uptake of [125 I]IMT increased rapidly during
the initial 10 min of incubation and then the intracellularconcentration of [125 I]IMT reached a steady state after afurther 5 min. Fig. 1C shows the contribution of transportsystem L to total uptake of [125I]IMT. Replacement of
sodium with choline reduced [125 I]IMT uptake only by asmall amount, suggesting that [125I]IMT is a substrate of an
Na+ -dependent transport system. Fig. 1D demonstrates
the concentration dependence of membrane transport of[125I]IMT in DLD-1 cells. The inset graph is a double
reciprocal plot for kinetic analysis. It revealed a high-affinity
apparent Michaelis constant of Km=78 μM, and a maximum
transport velocity (Vmax) of 333 pmol/106 cells per minute in
Na+-free buffer.
As shown in Fig. 2A and B, [125I]IMT uptake wasinhibited by neutral L- and D-amino acids (Cys, Leu, Ile, Phe,
Met, Tyr, His, Trp and Val) in DLD-1 cells. Ala, Ser and ThrTable 1
Primers for real-time PCR
GeneSequences (5′-3′)

Probe GenBank accession numberno.

Forward

Reverse

4F2hc

cagaagtggtggcacacg

gtaatcgagacgccccttc

81

LAT1
LAT2
LAT3
LAT4
ATA1
ATA2
ASCT1
MCT8
TAT1
B0AT1
ACTB
GAPDH

gtggaaaaacaagcccaagt
ttgccaatgtcgcttatgtc
gccctcatgattggctctta
caagtatggcccgaggaag
tctaatgggaaaggaagagacatt
cctatgaaatctgtacaaaagattgg
tttgcgacagcatttgctac
gctgccgttgctttcatt
ggtgtgaagaaggtttatctacagg
gcttcccctacctgtgtcag
ccaaccgcgagaagatga
agccacatcgctcagacac

cacctgcatgagcttctgac
ggagcttctctccaaaagtcac
ccggcatcgtagatcagc
caatcagcaagcaggaaacc
tgccaaaggtaactatcaccac
ttgtgtacccaatccaaaacaa
gcacttcatcatagagggaagg
tgaagtagcgcaggcttagg
agggccccaaagatgcta
ggatgaggaacgggatcat
ccagaggcgtacagggatag
gcccaatacgaccaaatcc

25
17
29
3
64
9
78
17
6
63
64
60

NM_001012661.1, NM_001012662.1, NM_002394.4, NM_001012663.1,
NM_001012664.1, NM_001013251.1
NM_003486.5
NM_012244.2, NM_182728.1
NM_003627.4
NM_152346.1
NM_030674.3, NM_001077484.1
NM_018976.3
NM_003038.2
NM_006517.2
NM_018593.3
NM_001003841.1
NM_001101.2
NM_002046.3

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N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 197–204

Fig. 1. Uptake mechanisms of 3-[125I]iodo-α-methyl-L-tyrosine ([125I]IMT), an amino acid transport marker, in colon cancer DLD-1 cells. (A) Growth curve of
DLD-1. (B) Time course study of [125I]IMT uptake into DLD-1 cells on the third or fourth day after inoculation. The [125I]IMT uptake was plotted against
incubation time. (C) Contributions of transport systems to total uptake of [125I]IMT. Closed and open squares represent uptake of [125I]IMT into DLD-1 cells in
Na+-containing or Na+-free medium, respectively, on the third or fourth day after inoculation. *Pb.0001; **Pb.005 vs. control. (D) Concentration dependence of
membrane transport of [125I]IMT in DLD-1 cells. The uptake was plotted against 3-iodo-α-methyl-L-tyrosine concentration. Uptake of [125I]IMT followed
Michaelis–Menten kinetics, with a Km of 78.0 μM and Vmax of 333 pmol/106 cells per minute in Na+-free buffer.

had weaker inhibitory effects on [125I]IMT uptake. Most of
the L-isomers demonstrated higher inhibitory effects than the+
D-isomers, particularly without Na . On the other hand, theinhibitory effects of D-Phe, D-Met, D-Tyr and D-Asn werehigher than those of the corresponding L-isomers in thepresence of sodium.
Fig. 3 shows the inhibitory effect of amino acids andamino acid-like drugs on [125I]IMT transport into DLD-1
cells in Na+-containing uptake medium.
The tested tyrosine analogues, tryptophan analogues,
L -phenylalanine and p-halogeno-L -phenylalanines, andgamma amino acids (including L-DOPA, DOPS, melphalan
and gabapentin) all inhibited [125I]IMT uptake, except for
L-tyrosine methyl ester and R(+)-/S(−)-baclofen. L-DOPA
inhibited [125I]IMT uptake to a greater extent than DOPS.
The substrates of systems ASC and A did not inhibit[125I]IMT uptake, except for L-serine and D/L-cysteine. Noneof the tested cationic or anionic amino acids inhibited[125I]IMT transport into DLD-1 cells.
The results of representative human neutral amino acidtransporter detection in DLD-1 cells by real-time PCR are
shown in Table 2. Expression of system L (4F2hc, LAT1 andLAT2), system A (ATA1, ATA2) and system ASC (ASCT1)
was strongly detected; system L (LAT3, LAT4) and MCT8
were weakly detected; and B0AT was not detected by realtime PCR.

4. Discussion
We characterized [125I]IMT uptake and uptake inhibitionby amino acids and amino acid-like drugs, using the humancolorectal adenocarcinoma cell line, DLD-1, which wasisolated by Dexter et al. [28]. Seeding of DLD-1 cells in agar
is known to result in clones of two distinct morphologies:
clone A and clone D [28]. We used DLD-1 cells 3 to 4 days
after inoculation and in semiconfluent logarithmic phase,before the appearance of these clones.
In the time course experiment, uptake of [125I]IMT into
DLD-1 cells increased rapidly during the initial 10 min of
incubation. [125I]IMT uptake by DLD-1 cells involves
primarily system L (about 90%), and [125I]IMT uptake wasinhibited by neutral L- and D-amino acids (Cys, Leu, Ile, Phe,
Met, Tyr, His, Trp and Val). This inhibition profile is similar

N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 197–204

201

Fig. 2. Effects of natural amino acids (1 mM) on [125I]IMT transport into DLD-1 cells on the third or fourth day after inoculation. (A) L-Amino acids. (B)125
D-Amino acids. Closed and open columns represent uptake of [I]IMT into DLD-1 cells in Na+-containing or Na+-free uptake medium, respectively.

to our recent report [29] suggesting that LAT1 is the maintransporter of [125I]IMT into DLD-1 cells. [125I]IMT appears
to be a substrate of an Na+-dependent transport system butwith only small affinity to such systems, because replacement of sodium with choline reduced [125 I]IMT uptake only
by a small amount. Ala, Ser and Thr had weaker inhibitory
effects on [125I]IMT uptake, suggesting [125 I]IMT affinity tosystem ASC [30]. With this partial [125 I]IMT uptake
inhibition caused by Ser and Cys, participation of LAT2
also appears likely, but LAT2 does not involve [125I]IMT
according to our oocyte experiments [31]. On the other hand,system A does not contribute to [125 I]IMT uptake into DLD1 cells because MeAIB, a system A-specific inhibitor, did
not demonstrate an inhibitory effect. In DLD-1 cells, the Km
and Vmax corresponding to system L (78 μM and 333 pmol/
106 cells per minute) are similar to those described elsewherein other cell lines [7]. These results do not contradict the
major neutral amino acid transporter gene expression profile

detected by real-time PCR: system L (4F2hc, LAT1), system
A (ATA1, ATA2) and system ASC (ASCT1).
LAT1 is strongly induced depending on the cellularconditions (i.e., cell activation, cellular proliferation andcellular nutrition) [18–20], which is of great interest to thosestudying not only cancer imaging [7] and therapeutics [21],but also a new class of target in immunosuppressant studies[22]. From this point of view, we conducted a study on
inhibition by neutral amino acids and amino acid-like drugsof [125 I]IMT transport into DLD-1 cells.
We observed an inhibitory effect on [125I]IMT transport
into DLD-1 cells by the following amino acid-like drugs orcompounds: DOPS, melphalan and p-halogeno-L-phenylalanines. The tested tyrosine analogues, tryptophan analogues
and phenylalanine analogues strongly inhibited [125I]IMTtransport into DLD-1 cells, except for L-tyrosine methyl
ester. These compounds are typical LAT1 selective substrates that are composed of large neutral alpha-amino

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N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 197–204

Fig. 3. Inhibitory effect of amino acids and amino acid-like drugs (1 mM) on [125I]IMT transport into DLD-1 cells in Na+-containing uptake medium on the thirdor fourth day after inoculation. (A, a) Substrates of neutral amino acid transport systems (tyrosine analogues, tryptophan analogues, L-phenylalanine andp-halogeno-L-phenylalanines, and gamma amino acids); (b) substrates of system ASC; (c) substrates of system A. (B) Substrates of cationic amino acidtransport systems. (C) Substrates of anionic amino acid transport systems. *Pb.001; **Pb.01 vs. control.

N. Shikano et al. / Nuclear Medicine and Biology 37 (2010) 197–204
Table 2
Results of real-time PCR
n=3

Target/GAPDH ratio×1000

Target/ACTB ratio×1000

GAPDH
ACTB

1000.00±58.19
1931.58±37.31

517.71±30.13
1000.00±19.32

4F2hc
LAT1
LAT2
LAT3
LAT4
ATA1
ATA2
ASCT1
MCT8
TAT1
B0AT1

109.47± 10.27
253.95±22.25
58.95±10.33
8.84±0.08
2.36±0.03
97.63±1.2164.53±10.52
21.11±4.05
3.66±0.18
2.12±0.16
0.00±0.00

56.68±5.32131.47±11.52
30.52±5.35
4.58±0.04
1.22±0.01
50.54±0.62
33.41±5.45
10.93±2.10
1.90±0.09
1.10±0.09
0.00±0.00

Values are shown as mean±S.D.

acids with branched or aromatic side chains, an alpha-aminogroup or alpha carboxyl group, except for L-tyrosine methyl
ester [29].
We recently reported that the introduction of hydrophobic
iodine into the benzene ring of L-tyrosine results in higher
transport affinity, and neither the alpha-methyl group
nor the size of the 3-iodinated-L-tyrosine residue was an
obstacle to transport via LAT1 [29]. In addition, thepossibility arises that beta-hydroxylation confers a reductionof transport affinity via LAT1, based on a comparison of thestructure and inhibitory effect of L-DOPA and DOPS on[125 I]IMT transport.
Gabapentin, which is not an alpha-amino acid, is a
substrate of LAT1, in addition to the alpha-amino acids [29].
Gabapentin, R(+)-baclofen and S(−)-baclofen are gamma
amino acid analogues, and, interestingly, we observed astatistically weaker inhibitory effect of R(+)-baclofen andS(−)-baclofen on [125I]IMT transport. Thus, whether gamma
amino acid analogue transport is involved in LAT1 may
depend on the structure of the group corresponding to theamino acid residue that combines with the beta-carbon.
With regard to the other amino acid transport systems,[125 I]IMT appears not to have an affinity for the cationic oranionic amino acid transport systems expressed in DLD-1, as
none of the tested cationic or anionic amino acids in thisstudy inhibited [125I]IMT transport into DLD-1 cells. To
date, four isoforms belonging to the human cationic aminoacid transporter family (CAT family) are known. Human
cationic amino acid transporter-1 is almost ubiquitously
expressed and probably the most important entity forsupplying cells with extracellular arginine, lysine andornithine [32]. It is expressed endogenously in DLD-1[32]. The expression profiles of the other cationic amino acidtransport systems, system b0,+ and system y+L, are notknown. Moreover, the expression in DLD-1 of the humananionic amino acid transport systems, system X−AG andsystem X−c , is also unknown. Numerous cell lines cultured invitro express xCT, a component of the system X−c transporter

203

family, and thus this system may be expressed in DLD-1 [33]as well, but [125 I]IMT may not have an affinity to xCT.
To study the characteristics of in vivo tumor imaging
probes, it is essential to consider not only the gene level andcell or tissue level argument, but also the whole-bodyargument. It is known that nude mice that receive a
subcutaneous inoculum of DLD-1 cells develop tumors
histologically similar to colonic adenocarcinomas in 10 to
14 days [34]. We plan to study improvements in tumor
imaging using colon cancer DLD-1 cells and DLD-1 tumorbearing mice.

5. Conclusions
We used real-time PCR to detect the gene expression ofsystem L (4F2hc, LAT1 and LAT2), system A (ATA1,
ATA2) and system ASC (ASCT1). Weaker expression ofsystem L (LAT3, LAT4) genes and the MCT8 gene was
detected, while that of B0AT was not detected. 125I-IMT
uptake into DLD-1 cells was inhibited by the substrates ofsystem L (main) and system ASC (minor), but not of system
A. In DLD-1 cells, the Km and Vmax corresponding to system
L (78 μM and 333 pmol/106 cells per minute) were observedin Na+-free buffer. Thus, [125I]IMT uptake in DLD-1 cells
involves primarily LAT1, and its substrates (including aminoacid-like drugs derived from tyrosine, tryptophan andphenylalanine) inhibit [125 I]IMT uptake. Moreover, whether
gamma amino acid analogue transport is involved in LAT1
depends on the structure of the group corresponding to theamino acid residue. The possibility exists that betahydroxylation of L-DOPA confers reduction of affinity to[125I]IMT transport via LAT1.
Acknowledgments
We wish to thank Natsumi Mibuka, Yuki Kitamura,
Yuzou Taguchi, Miho Aoyama, Hironobu Sakai, Misako
Nozaki, Yuko Fujisaku, Mariko Maruyama and Takayoshi
Miyakawa of Ibaraki Prefectural University for their
excellent technical assistance.
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